Pharmaceutical Composition Comprising Cyclic Somatostatin Analogues

ABSTRACT

The present invention describes parenteral pharmaceutical compositions comprising a somatostatin analogue and novel somatostatin analogues.

This application is a divisional of U.S. application Ser. No.12/359,527, filed Jan. 26, 2009, which is a continuation of U.S.application Ser. No. 10/560,751, which is a 371 of PCT/EP2004/006794filed Jun. 23, 2004, which claims benefit of Great Britain ApplicationNo. 0314695.8 filed Jun. 24, 2003 and Great Britain Application No.0325388.7 filed Oct. 30, 2004, which in their entirety are hereinincorporated by reference.

The present invention relates to parenteral pharmaceutical compositionscomprising a somatostatin analogue and to novel somatostatin analogues.

Somatostatin is a tetradecapeptide having the structure

Since the isolation and characterization of somatostatin, an extensivesearch for more potent and more stable analogues has continued.

Somatostatin analogues have been described e.g. in WO 97/25977. Saidsomatostatin analogues comprise the amino acid sequence of formula I

-(D/L)Trp-Lys-X₁—X₂—  I

wherein X₁ is a radical of formula (a) or (b)

wherein R₁ is optionally substituted phenyl,

R₂ is —Z₁—CH₂—R₁, —CH₂—CO—O—CH₂—R₁,

wherein Z₁ is O or S, and

X₂ is an α-amino acid having an aromatic residue on the C_(α) sidechain, or an amino acid unit selected from Dab, Dpr, Dpm,His,(Bzl)HyPro, thienyl-Ala, cyclohexyl-Ala and t-butyl-Ala, the residueLys of said sequence corresponding to the residue Lys⁹ of the nativesomato-statin-14.

By somatostatin analogue as used herein is meant a straight-chain orcyclic peptide derived from that of the naturally occurringsomatostatin-14, comprising the sequence of formula I and whereinadditionally one or more amino acid units have been omitted and/orreplaced by one or more other amino acid radical(s) and/or wherein oneor more functional groups have been replaced by one or more otherfunctional groups and/or one or more groups have been replaced by one orseveral other isosteric groups. In general the term covers all modifiedderivatives of the native somatostatin-14 comprising the above sequenceof formula I which have binding affinity in the nM range to at least onesomatostatin receptor subtype as defined hereinafter.

Preferred are somatostatin analogues in which the residues at positions8 through 11 of the somatostatin-14 are represented by the sequence offormula I as defined above.

More preferred are somatostatin analogues as disclosed above comprisinga hexapeptide unit, the residues at positions 3 through 6 of saidhexapeptide unit comprising the sequence of formula I. Even morepreferably the residues at positions 1 and 2 of the hexapeptide unit ofthe somatostatin hexapeptide may be any of those as known in the art,e.g. as disclosed by A. S. Dutta in Small Peptides, Vol.19, 292-354,Elsevier, 1993, or as substituents for, Phe⁶ and/or Phe⁷ ofsomatostatin-14.

Even more preferred are cyclic somatostatin hexapeptides, e.g. cyclicsomatostatin hexapeptides comprising a hexapeptide unit numbered from 1to 6, the residues at positions 3 through 6 of said hexapeptide unithaving the amino sequence of formula I as indicated above, e.g. acompound of formula Ia

wherein X₁ and X2 are as defined above,

A is a divalent residue selected from Pro,

wherein R₃ is NR₈R₉—C₂₋₆alkylene, guanidino-C₂₋₆alkylene orC₂₋₆alkylene-COOH, R_(3a) is H, C₁₋₄alkyl or has independently one ofthe significances given for R₃, R_(3b) is H or C₁₋₄alkyl, R_(a) is OH orNR₅R₆, R_(b) is —(CH₂)₁₋₃— or —CH(CH₃)—, R₄ is H or CH₃, R_(4a) isoptionally ring-substituted benzyl, each of R₅ and R₆ independently isH, C₁₋₄alkyl, ω-amino-C₁₋₄alkylene, ω-hydroxy-C₁₋₄alkylene or acyl, R₇is a direct bond or C₁₋₆alkylene, each of R₈ and R₉ independently is H,C₁₋₄alkyl, ω-hydroxy-C₂₋₄alkylene, acyl or CH₂OH—(CHOH)_(c)—CH₂— whereinc is 0, 1, 2, 3 or 4, or R₈ and R₉ form together with the nitrogen atomto which they are attached a heterocyclic group which may comprise afurther heteroatom, and R₁₁ is optionally ring-substituted benzyl,—(CH₂)₁₋₃—OH, CH₃—CH(OH)— or —(CH₂)₁₋₅—NR₅R₆, and ZZ_(a) is a natural orunnatural a-amino acid unit.

Particularly preferred are compounds of formula II

wherein the configuration at C-2 is (R) or (S) or a mixture thereof, and

wherein R is NR₁R₂—C₂₋₆alkylene or guanidine-C₂₋₆alkylene, and each ofR₁ and R₂ independently is H or C₁₋₄alkyl,

in free form, in salt form or protected form.

Preferably R is NR₁R₂—C₂₋₆alkylene. Preferred compounds of formula IIare the compounds wherein R is 2-amino-ethyl, namelycyclo[{4-(NH₂—C₂H₄—NH—CO—O-)Pro}-Phg-DTrp-Lys-Tyr(4-Bzl)-Phe] (referredherein to as Compound A) andcyclo[{4-(NH₂—C₂H₄—NH—CO—O-)Pro}-DPhg-DTrp-Lys-Tyr(4-Bzl)-Phe], in freeform, salt form or protected form. Phg means —HN—CH(C₆H₅)—CO— and Bzlmeans benzyl.

These compounds in free form, salt form or protected form are referredto hereinafter as “compounds of the invention”.

Due to proteolytic degradation of the somatostatin analogues of thepresent invention, systemic delivery, e.g. parenteral administration, ishighly desirable. However, parenteral administration may be very painfulat the site of administration, especially in repeated administration.

It has now been found that parenteral compositions comprising a compoundof the invention, and tartaric acid show particularly interestingproperties, e.g. good tolerability and high stability.

A compound of the invention in protected form corresponds to asomatostatin analogue wherein at least one of the amino groups isprotected and which by deprotection leads to a compound of formula II,preferably physiologically removable. Suitable amino protecting groupsare e.g. as disclosed in “Protective Groups in Organic Synthesis”, T. W.Greene, J. Wiley & Sons NY (1981), 219-287, the contents of which beingincorporated herein by reference. Example of such an amino protectinggroup is acetyl.

A compound of the invention may exist e.g. in free or salt form. Saltsinclude acid addition salts with e.g. inorganic acids, polymeric acidsor organic acids, for example with hydrochloric acid, acetic acid,lactic acid, aspartic acid, benzoic acid, succinic acid or pamoic acid.Acid addition salts may exist as mono- or divalent salts, e.g. dependingwhether 1 or 2 acid equivalents are added. Preferred salts are thelactate, aspartate, benzoate, succinate and pamoate including mono- anddi-salts, more preferably the aspartate di-salt and the pamoatemonosalt.

The compounds of the invention may be prepared in accordance withconventional methods.

In a first aspect, the present invention provides a parenteralcomposition comprising a compound of the invention and tartaric acid.

According to the invention, typically the concentration of the compoundof the invention in the composition of the invention is from about 0.05to about 1 mg per ml composition, particularly 0.1 to 1 mg/ml.

Conveniently, the ratio of the compound of the invention (amountcorresponding to free form) to tartaric acid is about 0.001 to about 2weight in weight, preferably about 0.05 to about 0.6.

The amount of the compound of the invention in the composition of theinvention is from about 0.005% to about 0.1% based on a total weight ofthe formulation.

Preferably, the tartaric acid is in fine crystalline form. Morepreferably, crystalline D(−) or L(+) tartaric acid is used. The amountof tartaric acid is preferably from about 0.01% to about 1.5% w/w of theformulation, preferably about 0.01% to about 0.3%, more preferably about0.15%. Preferably, the molarity of tartaric acid in the finalcomposition is about 10 mM.

In accordance with the present invention, in addition to the tartaricacid and a compound of the invention, the pharmaceutical compositionpreferably comprises also a basic component selected and added to thecomposition in such a way that the pH of the tartaric acid bufferedpharmaceutical composition is adjusted to a pH of about 4 to about 4.5,preferably about 4.2.

Preferably, the basic component is a base, e.g. sodium hydroxide orpotassium hydroxide, or a basic salt e.g. sodium hydrogen carbonate,sodium carbonate, potassium hydrogen carbonate, or potassium carbonate.Preferably, the basic component is added in such an amount that theresulting pharmaceutical composition has a pH buffered as indicatedabove.

Preferably, the pharmaceutical composition of the invention is waterbased.

The compositions of the invention may further comprise a tonicity agentsuch as mannitol, sodium chloride, glucose, dextrose, sucrose, orglycerins. Preferably, the tonicity agent is mannitol.

The amount of tonicity agent is chosen to adjust the isotonicity of thecomposition of the invention, e.g. mannitol preferably may be from about1% to about 5% by weight of the composition, preferably about 4.95%.Conveniently, mannitol is present in a ratio mannitol to tartaric acidof about 20 to about 40, preferably about 33.

The compositions of the invention may contain additional excipientscommonly employed in parenteral compositions in order to provide therequired stability and therapeutic efficacy. Excipients may include e.g.an antioxidant or a preserving agent.

Antioxidants may be employed to protect the active agent from oxidativedegradation particularly under the accelerated conditions of thermalsterilisation. Antioxidants may be selected from any of those compoundsknown in the art. Similarly, the amount of antioxidant employed can bedetermined using routine experimentation. Preferably, the compositionsof the invention do not contain an antioxidant.

A preserving agent, e.g. phenol, may preferably be added to thecomposition when it is formulated as multidose vials, cartridges orsyringes. Preferably, the compositions of the invention do not contain apreserving agent.

Reference is made to the extensive literature on the subject for theseand other excipients and procedures mentioned herein, see in particularHandbook of Pharmaceutical Excipients, Second Edition, edited by AinleyWade and Paul J. Weller, American Pharmaceutical Association,Washington, USA and Pharmaceutical Press, London; and Lexikon derHilfsstoffe für Pharmazie, Kosmetik and angrenzende Gebiete edited by H.P. Fiedler, 4th Edition, Editio Cantor, Aulendorf and earlier editionswhich are incorporated herein by reference.

Preferably, the composition of the invention contains as activeingredient only compound of the invention, e.g. a compound of formulaII, e.g. Compound A.

Procedures which may be used to prepare the compositions of theinvention may be conventional or known in the art or based on suchprocedures e.g. those described in L. Lachman et al. The Theory andPractice of Industrial Pharmacy, 3rd Ed, 1986, H. Sucker et al,Pharmazeutische Technologie, Thieme, 1991, Hager's Handbuch derpharmazeutischen Praxis, 4th Ed. (Springer Verlag, 1971) and Remington'sPharmaceutical Sciences, 13th Ed., (Mack Publ., Co., 1970) or latereditions.

Typically, the compound of the invention, the tartaric acid andoptionally the other ingredients as mentioned in the desired amount aredissolved in an aqueous solvent, preferentially in water for injection,and the pH is adjusted with the base. The resulting solution may then bediluted with water to make it up to the final desired volume. Theresulting solution may be filtered through a sterile filter, e.g. aMillipak® filter. Preferably, during above preparation oxygen (air) isdisplaced from contact with the solution of the compound of theinvention. This is usually carried out by purging with, e.g. nitrogen, acontainer holding the solution. The pharmaceutical composition may bepacked under carbon dioxide or other inert gas to prevent degradation,preferably under carbon dioxide, e.g. charged into vials, e.g. glassvials, ampoules, e.g. glass ampoules, or syringes, e.g. prefilledsyringes, and steam or heat sterilized.

The solution may be freeze-dried by a conventional method under asepticconditions to give a powder for injection which may be used toreconstitute the desired solution for parenteral administration shortlybefore administration by mixing the powder with the desired amount ofsolvent e.g. with water for injection.

Alternatively, the present invention provides in another aspect acomposition for parenteral administration buffered at a pH of about 4 toabout 4.5, preferably about 4.2, and comprising as active ingredientcompound A or a pharmaceutically acceptable salt thereof, e.g. alactate, mono- or di-aspartate, succinate, preferably a aspartatedi-salt.

These compositions may comprise the same components as described abovefor compositions comprising tartaric acid wherein the tartaricacid/tartrate is replaced by another buffer such as acetate/acetic acid,lactate/lactic acid, and Glycin/HCl.

The compositions of the invention are useful

a) for the prevention or treatment of disorders with an aetiologycomprising or associated with excess GH-secretion and/or excess of IGF-1e.g. in the treatment of acromegaly as well as in the treatment of typeI or type II diabetes mellitus, especially complications thereof, e.g.angiopathy, diabetic proliferative retinopathy, diabetic macular edema,nephropathy, neuropathy and dawn phenomenon, and other metabolicdisorders related to insulin or glucagon release, e.g. obesity, e.g.morbid obesity or hypothalamic or hyperinsulinemic obesity,

b) in the treatment of enterocutaneous and pancreaticocutaneous fistula,irritable bowel syndrom, inflammatory diseases, e.g. Grave's Disease,inflammatory bowel disease, psoriasis or rheumatoid arthritis,polycystic kidney disease, dumping syndrom, watery diarrhea syndrom,AIDS-related diarrhea, chemotherapy-induced diarrhea, acute or chronicpancreatitis and gastrointestinal hormone secreting tumors (e.g. GEPtumors, for example vipomas, glucagonomas, insulinomas, carcinoids andthe like), lymphocyte malignancies, e.g. lymphomas or leukemias,hepatocellular carcinoma as well as gastrointestinal bleeding, e.g.variceal oesophagial bleeding,

c) for the prevention or treatment of angiogenesis, inflammatorydisorders as indicated above including inflammatory eye diseases,macular edema, e.g. cystoid macular edema, idiopathic cystoid macularedema, exudative age-related macular degeneration, choroidalneovascularization related disorders and proliferative retinopathy,

d) for preventing or combating graft vessel diseases, e.g. allo- orxenotransplant vasculo-pathies, e.g. graft vessel atherosclerosis, e.g.in a transplant of organ, e.g. heart, lung, combined heart-lung, liver,kidney or pancreatic transplants, or for preventing or treating veingraft stenosis, restenosis and/or vascular occlusion following vascularinjury, e.g. caused by catherization procedures or vascular scrapingprocedures such as percutaneous transluminal angioplasty, lasertreatment or other invasive procedures which disrupt the integrity ofthe vascular intima or endothelium,

e) for treating somatostatin receptor expressing or accumulating tumorssuch as pituitary tumors, e.g. Cushing's Disease,gastro-enteropancreatic, carcinoids, central nervous system, breast,prostatic (including advanced hormone-refractory prostate cancer),ovarian or colonic tumors, small cell lung cancer, malignant bowelobstruction, paragangliomas, kidney cancer, skin cancer, neuroblastomas,pheochromocytomas, medullary thyroid carcinomas, myelomas, lymphomas,Hodgkins and non-Hodgkins lymphomas, bone tumours and metastasesthereof, as well as autoimmune or inflammatory disorders, e.g.rheumatoid arthritis, Graves disease or other inflammatory eye diseases.

Preferably, the compositions of the invention are useful in thetreatment of acromegaly and cancer, e.g. Cushing's Disease.

The activity and characteristics of the compositions of the inventionmay be indicated in standard clinical or animal tests.

Appropriate dosage of the composition of the invention will of coursevary, e.g. depending on the condition to be treated (for example thedisease type or the nature of resistance), the drug used, the effectdesired and the mode of administration.

When given continuously, an effective amount of drug may be given in twoor three doses spread over time such as by parenteral administration,e.g. intravenous drip, intramuscular or subcutaneous injection(s), orsubcutaneous infusion, e.g. continuous subcutaneous infusion, preferablysubcutaneous injection or infusion, with the total daily dose beingspread across the portion or the entire administration period. Whengiven by subcutaneous injection, it is most preferably administered from3 times per week up to 3 times a day, preferably twice a week up to onceor twice daily. A compound of the invention may also be administered inthe form of e.g. a subcutaneous bolus injection.

The composition of the invention preferably is suitable for subcutaneousadministration.

After injection, the composition of the invention is locally welltolerated. Particularly, the parenteral administration of a compositionof the invention, e.g. subcutaneous injection, leads to mild to noburning sensation at the injection site.

In addition to the good local tolerance after injection, the compositionof the invention exhibits good stability characteristics. For example,less than 2.5% of degradation products were found after 4 weeks storageat 60° C. For example, if stored with light protection at 2° C. to 8°C., the compositions of the invention are stable over 24 months.Particularly good stability may be observed with the diaspartate salt ofCompound A.

In general, satisfactory results are obtained on administration, e.g.subcutaneous administration, at dosages on the order of from about 0.01to about 1.2 mg, preferably from about 0.1 to about 0.6 mg of thecompound of the invention per injection or about 0.001 to about 0.009 mgper kg animal body weight per day, administered once or in divided dosesup to 4 times per day. Suitable daily dosages for patients are thus inthe order of about 0.1 mg to about 0.6 mg of a compound of theinvention, e.g. a compound of formula II, e.g. Compound A.

The following Examples serve to illustrate the compositions of theinvention.

EXAMPLES 1 to 7

Tartaric acid and mannitol are dissolved in water for injection, whilethe solution is purged with nitrogen. Then diaspartate salt of compoundA is added, the solution is adjusted with sodium hydroxide to pH 4.20and water for injection up to 1.0 ml is added. Under aseptic conditions,the solution is filtered through a Millipak-200® sterile filter with apore size ≦0.22 μm, filled into ampoules and sterilized by autoclaving.

Ex 1 Ex 2 Ex 3 Ex 4 diaspartate salt of Compound A 0.251 0.315 0.3760.472 (corresponding amount of Compound A) (0.200) (0.251) (0.300)(0.376) tartaric acid crystalline 1.501 1.501 1.501 1.501 mannitol49.500 49.500 49.500 49.500 sodium hydroxide 1N for injection ad pH 4.20ad pH 4.20 ad pH 4.20 ad pH 4.20 water for injection ad 1 ml ad 1 ml ad1 ml ad 1 ml Ex 5 Ex 6 Ex 7 diaspartate salt of Compound A 0.752 0.7861.129 (corresponding amount of Compound A) (0.600) (0.627) (0.900)tartaric acid crystalline 1.501 1.501 1.501 mannitol 49.500 49.50049.500 sodium hydroxide 1N for injection ad pH 4.20 ad pH 4.20 ad pH4.20 water for injection ad 1 ml ad 1 ml ad 1 ml

In yet another aspect, the present invention provides novel compounds offormula III

wherein R is NR₁R₂—C₂₋₆alkylene or guanidine-C₂₋₆alkylene, and

each of R₁and R₂ independently is H or C₁₋₄alkyl,

in free form, in salt form or complex form, or in protected form.

Preferably R is NR₁R₂—C₂₋₆alkylene. A preferred compound of formula IIIis the compound wherein R is 2-amino-ethyl, also calledcyclo[{4-(NH₂—C₂H₄—NH—CO—O-)Pro}-DPhg-DTrp-Lys-Tyr(4-Bzl)-Phe], andreferred herein to as Compound B,

in free form, in salt or complex form or in protected form. Phg and Bzlare as defined above.

These compounds in free form, in salt form or complex form, or inprotected form are referred hereinafter as “novel compounds of theinvention”.

A compound of formula III, e.g. Compound B, in protected formcorresponds to above molecule wherein at least one of the amino groupsis protected and which by deprotection leads to a compound of formulaIII, preferably physiologically removable. Suitable amino protectinggroups are e.g. as disclosed in “Protective Groups in OrganicSynthesis”, T. W. Greene, J. Wiley & Sons NY (1981), 219-287, thecontents of which being incorporated herein by reference. Example ofsuch an amino protecting group is acetyl.

When a compound of formula III, e.g. Compound B, exists in complex form,it may conveniently be a compound of formula III bearing a chelatinggroup on the side chain amino group of Pro and complexed with adetectable or radiotherapeutic element. Compound B bearing a chelatinggroup is referred to hereinto as conjugated Compound B.

Examples of chelating groups include e.g. those derived frompoly-aminopolycarboxylic acids or anhydrides, e.g. those derived fromnon cyclic ligands e.g. diethylene triamine pentaacetic acid (DTPA),ethylene glycol-0,0′-bis(2-aminoethyl)-N,N,N′,N′-tetraacetic acid(EGTA), N,N′-bis(hydroxybenzyl)ethylenediamine-N,N′-diacetic acid (HBED)and triethylene-tetramine hexaacetic acid (TTHA), those derived fromsubstituted DTPA, e.g. p-isothio-cyanato-benzyl-DTPA, those derived frommacrocyclic ligands, e.g.1,4,7,10-tetra-azacyclododecane-N,N′,N″,N″′-tetraacetic acid (DOTA),1,4,8,11-tetraazacyclotetra-decane-N,N′,N″,N′″-tetraacetic acid (TETA),or 1,4,7,10-tetraazacyclotridecane-N,N′,N″,N″′-tetraacetic acid (TITRA).

The chelating group may be attached either directly or through a spacerto the side chain amino group of Pro. Suitable spacers include thoseknown in the art, e.g. as disclosed in GB-A-2,225,579, for example thedivalent residue of an amino-carboxylic acid, for example β-Ala or adivalent residue derived from 6-amino-caproic acid.

Preferred chelating groups are those derived from DTPA, DOTA or TETA.Chelating groups derived from DTPA or DOTA are most preferred.

By detectable element is meant any element, preferably a metal ion whichexhibits a property detectable in vivo diagnostic techniques, e.g. ametal ion which emits a detectable radiation or a metal ion which iscapable of influencing NMR relaxation properties. By radiotherapeuticelement is meant any element which emits a radiation having a beneficialeffect on the conditions to be treated.

Suitable elements include for example heavy elements or rare earth ions,e.g. as used in CAT scanning (Computer axial tomography), paramagneticions, e.g. Gd³⁺, Fe³⁺, Mn²⁺ and Cr²⁺, fluorescent metal ions, e.g. Eu³⁺,and radionuclides, e.g. a radiolanthanide, particularly a γ-emittingradionuclide, β-emitting radionuclide, α-emitting radionuclide,Auger-e⁻-emitting radionuclide or a positron-emitting radionuclide e.g.⁶⁸Ga, ¹⁸F or ⁸⁶Y.

Suitable γ-emitting radionuclides include those which are useful indiagnostic techniques. The γ-emitting radionuclides advantageously havea half-life of from 1 hour to 40 days, preferably from 5 hours to 4days, more preferably from 12 hours to 3 days. Examples areradioisotopes from Gallium, Indium, Technetium, Ytterbium, Rhenium,Terbium, Lutetium, Thallium and Samarium e.g. ⁶⁷Ga, ¹¹¹In, ^(99m)Tc,¹⁶¹Tb, ¹⁶⁹Yb, ¹⁸⁶Re or ¹⁷⁷Lu.

Suitable β-emitting radionuclides include those which are useful inradiotherapeutic applications, for example ⁹⁰Y, ⁶⁷Cu, ¹⁸⁶Re, 188Re,¹⁶⁹Er, ¹²¹Sn, ¹²⁷Te, ¹⁷⁷Lu, ¹⁴³Pr, ¹⁹⁸Au, ¹⁰⁹Pd, ¹⁶⁵Dy, ¹⁴²Pr or ¹⁵³Sm.

Suitable α-emitting radionuclides are those which are used intherapeutic treatments, e.g. ²¹¹At, ²¹²Bi or ²⁰¹Tl.

Compounds of formula III, e.g. Compound B, may exist e.g. in free orsalt form. Salts include acid addition salts with e.g. inorganic acids,polymeric acids or organic acids, for example with hydrochloric acid,acetic acid, lactic acid, aspartic acid, benzoic acid, succinic acid orpamoic acid. Acid addition salts may exist as mono- or divalent salts,e.g. depending whether 1 or 2 acid equivalents are added to the CompoundB in free base form. Preferred salts are the lactate, aspartate,benzoate, succinate and pamoate including mono- and di-salts, morepreferably the aspartate di-salt and the pamoate monosalt.

The conjugated compounds of formula III, e.g. conjugated Compound B, mayadditionally exist in salt forms obtainable with the carboxylic acidgroups when present in the chelating group, e.g. alkali metal salts suchas sodium or potassium, or substituted or unsubstituted ammonium salts.

The present invention also includes a process for the production of acompound of formula III, e.g. Compound B. It may be produced in analogyto known methods, for example:

a) cyclising a linear peptide in protected, polymer-bound or unprotectedform in such a way that a compound of formula III, e.g. Compound B, isobtained and then optionally removing the protecting group(s),

b) to produce a conjugated compound of formula III, e.g. conjugatedCompound B, linking together a chelating group and the compound offormula III, e.g. Compound B, in protected or unprotected form and thenoptionally removing the protecting group, and recovering the compound offormula III, e.g. Compound B, or a conjugated compound of formula III,e.g. conjugated Compound B thus obtained, in free form, in salt form oroptionally complexed with a detectable or radiotherapeutic element.

It is generally not critical which amino acid is selected to be at theC-terminal position to start the peptide chain since the linear peptidewill be cyclized, provided only that the sequence of amino acids in thelinear peptide corresponds to that in the desired compound of formulaIII, e.g. Compound B. However there may be other factors which mayprefer one starting amino acid over another. When a compound of formulaIII, e.g. Compound B, is prepared by solid phase synthesis, the firstamino-acid is preferably attached to the resin, e.g. a commerciallyavailable polystyrene-based resin, through a suitable linker, e.g. alinker which is cleavable under mild conditions to keep the side chainprotection intact, e.g. SASRIN or an optionally substituted trityl basedlinker, for example 4-(hydroxy-diphenyl-methyl)-benzoic acid wherein onethe phenyl groups may optionally be substituted e.g. by Cl. The buildingup of the desired peptide chain may be effected in conventionnal manner,e.g. using amino-acid units wherein the terminal amino group isFmoc-protected, the side chain amino groups where present beingprotected with a different amino protecting group, e.g. Boc or CBO.Preferably the linear peptide is cyclized in such a way to produce abond between Tyr(4-Bzl)-OH and Phe, e.g.Phe-{4-(NHR₁-C₂H₄—NH—CO—O-)Pro}-DPhg-DTrp(R₂)-Lys(ε-NHR₃)-Tyr(4-Bzl)-OHor a functional derivative thereof, wherein each of R₁, R₂ and R₃ is anamino protecting group. The cyclisation step a) may conveniently beperformed according to known method, e.g. via an azide, an active ester,a mixed anhydride or a carbodiimide. Thereafter the protecting groupsare removed, e.g. by cleavage e.g. with trifluoroacetic or byhydrogenation.

The cyclisation of the peptide may also be performed directly on thesolid support, the first amino acid being in a Nα- and C-terminalprotected form and attached through a side chain, e.g. ε-amino functionof Lys or by backbone anchoring. The linear sequence is then synthesizedfollowing standard solid phase synthesis (SPPS) procedures. Aftercleavage of the C-terminal protection the peptide is cyclized e.g. asdescribed above. Thereafter the cyclic peptide is cleaved from the resinand deprotected.

If desired, the lateral chain present on Pro may be introduced on theamino acid prior to or after the peptide cyclisation step a). Thus, Proas a starting amino-acid or a starting linear or cyclic peptide whereinin each case Pro is ring-substituted by OH, may be converted to providea compound of formula III, e.g. Compound B, or the desired Pro unit orthe corresponding linear peptide, respectively, wherein Pro issubstituted by NHR₁—C₂H₄—NH—CO—O—.

The complexation of a conjugated compound of formula III, e.g.conjugated Compound B, may be performed by reacting the conjugatedcompound of formula III, e.g. the conjugated Compound B, with acorresponding detectable or radiotherapeutic element yielding compound,e.g. a metal salt, preferably a water-soluble salt. The reaction may becarried out by analogy with known methods, e.g. as disclosed in Perrin,Organic Ligand, Chemical Data Series 22. NY Pergamon Press (1982); inKrejcarit and Tucker, Biophys. Biochem. Res. Corn. 77: 581 (1977) and inWagner and Welch, J. Nucl. Med. 20: 428 (1979).

The following examples are illustrative of the novel compounds of theinvention of formula III. All temperatures are in ° C.

Abbreviations:

-   AcOH=acetic acid-   Boc=tert.-butoxy-carbonyl-   Bzl=benzyl-   CBO=carbobenzoxy-   DIPCI=N,N′-diisopropylcarbodiimide-   DIPEA=diisopropylethylamine-   DMF=dimethylformamide-   DPPA=diphenyiphosphorylazide-   Fmoc=fluorenylmethoxycarbonyl-   HOBT=1-hydroxybenzotriazole-   Osu=N-hydroxysuccinimide-   TFA=trifluoroacetic acid-   THF=tetrahydrofuran

EXAMPLE 8 Cyclo[{4-(NH₂—C₂H₄—NH—CO—O-)Pro}-DPhg-DTrp-Lys-Tyr(4-Bzl)-Phe]

a) Synthesis of Fmoc-Pro(4-OCO—NH—CH₂—CH₂—NH-Boc)-OH

L-hydroxyproline methylester hydrochloride is reacted with Fmoc-OSu inaqueous 1.0 N sodium carbonate/THF at room temperature. After completionof the reaction, Fmoc-Pro(4-OH)—OMe is isolated by precipitation.Fmoc-Pro(4-OH)—OMe is then added dropwise into a solution oftrisphosgene (0.6 eq.) in THF to give a chlorocarbonate intermediate.After 1 h dimethylaminopyridine (1.0 eq.) and N-Boc-diaminoethane (6.0eq.) are added and the reaction is stirred at room temperature. Aftercompletion of the reaction, the solvent is removed in vacuo and theresulting Fmoc-Pro(4-OCO—NH—CH₂—CH₂—NH-Boc)-OMe is extracted from a twophase system of ethyl acetate/0.1 M HCl to give crude product (MH⁺=554)which is purified by crystallization from ethyl acetate. The methylester is then cleaved to the free acid by treatment with 1N NaOH indioxane/water and the product Fmoc-Pro(4-OCO—NH—CH₂—CH₂—NH-Boc)-OH ispurified on silica gel, [(M+Na)]⁺=562).

b)H-Phe-Pro(4-OCO—NH—CH₂—CH₂—NH-Boc)-DPhg-DTrp(Boc)-Lys(Boc)-Tyr(Bzl)-OH

Commercially available Fmoc-Tyr(Bzl)-O—CH₂—Ph(3-OCH₃)—O—CH₂-Polystyreneresin (SASRIN-resin, 2.4 mM) is used as starting material and carriedthrough a standard protocol consisting of repetitive cycles ofNα-deprotection (Piperidine/DMF, 2:8), repeated washings with DMF andcoupling (DIPCI: 4.8 mM/HOBT: 6 mM, DMF). The following aminoacid-derivatives are sequentially coupled: Fmoc-Lys(Boc)-OH,Fmoc-DTrp(Boc)-OH, Fmoc-DPhg-OH, Fmoc-Pro(4-OCO—NH—CH₂—CH₂—NH-Boc)-OH,Fmoc-Phe-OH. Couplings (2 eq. amino acids) are continued or repeateduntil completion, i.e. until complete disappearance of residual aminogroups which is monitored by a negative ‘Kaiser’ Ninhydrin test. Beforecleavage of the completely assembled protected linear peptide from itsresin support the Nα-Fmoc protection from the last residue is removed.

c)H-Phe-Pro(4-OCO—NH—CH₂—CH₂—NH-Boc)-DPhg-DTrp(Boc)-Lys(Boc)-Tyr(Bzl)-OH

After washings with CH₂Cl₂, the peptide-resin is transferred into acolumn or a stirred suction filter and the peptide fragment is cleavedand eluted with a short treatment with 2% TFA in CH₂Cl₂ within 1 h. Theeluate is immediately neutralized with a saturated NaHCO₃ solution. Theorganic solution is separated and evaporated and the side chainprotected precursor (MH⁺=1366) is cyclized without further purification.

d) cyclo[-Pro(4-OCO—NH—CH₂—CH₂—NH₂)-DPhg-DTrp-Lys-Tyr(Bzl)-Phe-],trifluoroacetate

The above linear fragment is dissolved in DMF (4 mM), cooled to minus 5°C. and treated with 2 eq. DIPEA then 1.5 eq. of DPPA and stirred untilcompletion (ca. 20 h) at 0-4° C. The solvent was almost completelyremoved in vacuo; the concentrate is diluted with ethyl acetate, washedwith NaHCO₃, water, dried and evaporated in vacuo.

For deprotection the residue is dissolved at 0° C. in TFA/H₂O 95:5 (ca.50 mM) and stirred in the cold for 30 min. The product is thenprecipitated with ether containing ca. 10 eq. HCl, filtered, washed withether and dried. In order to completely decompose remaining Indole-Ncarbaminic acid the product is dissolved in 5% AcOH and lyophilizedafter 15 h at ca. 5° C. Preparative RP-HPLC is carried out on a C-18 10μm STAGROMA column (5-25 cm) using a gradient of 0.5% TFA to 0.5% TFA in70% acetonitrile. Fractions containing the pure title compound arecombined, diluted with water and lyophilized. The lyophilisate isdissolved in water followed by precipitation with 10% Na₂CO₃ in water.The solid free base is filtered of, washed with water and dried invacuum at room temperature. The resulting white powder is directly usedfor the different salts.

EXAMPLE 9 Cyclo[{4-(NH₂—C₂H₄—NH—CO—O-)Pro}-DPhg-DTrp-Lys-Tyr(4-Bzl)-Phe]in salt form

a. Acetate

Conversion to the acetate salt form is carried out using an ion-exchangeresin (e.g. AG 3-X4). MS (ESI): m/z 524.5 [M+2H]²⁺

[α]_(D) ²⁰=−41.6°; c=0.56; AcOH 95%; T=20C; 589.3 nm

b. Aspartate

Conversion to the mono- or di-aspartate is obtained by reacting 1equivalent of the compound of Example 8 with 1 or 2 equivalent ofaspartic acid in a mixture of acetonitrile/water 1:3. The resultingmixture is frozen and lyophilized.

The di-aspartate may also be obtained by dissolving the compound ofExample 8 in water/acetonitrile 4:1, filtering, loading on a anion-exchange resin, e.g. BioRad AG4X4 column, and eluting withwater/acetonitrile 4:1. The eluate is concentrated, frozen andlyophilized.

c. Benzoate

Conversion to the benzoate may be obtained by dissolving the compound ofExample 8 with 2 equivalents of benzoic acid in a mixture ofacetonitrile/water 1:2. The resulting mixture is frozen and lyophilized.

d. Pamoate

1 equivalent of the compound of Example 8 is dissolved together with 1equivalent of embonic acid in a mixture of acetonitrile/THF/water 2:2:1.The resulting mixture is frozen and lyophilized.

EXAMPLE 10Cyclo[{4-(DOTA-NH—C₂H₄—NH—CO—O)Pro}-DPhg-DTrp-Lys-Tyr(4-Bzl)-Phe

a) cyclo[-Pro(4-OCO—NH—CH₂—CH₂—NH₂)-DPhg-DTrp-Lys(Cbo)-Tyr(Bzl)-Phe-],trifluoroacetate

The compound is synthesised in the same way likecyclo[-Pro(4-OCO—NH—CH₂—CH₂—NH₂)-DPhg-DTrp-Lys(Cbo)-Tyr(Bzl)-Phe],trifluoroacetate by using Fmoc-Lys(Cbo)-OH instead of Fmoc-Lys(Boc)-OH.

b) 400 mg commercially available DOTA×2H₂O (SYMAFEX—France) is dissolvedin 20 ml water. After addition of 20 ml DMF, 170 mgcyclo[-Pro(4-OCO—NH—CH₂—CH₂—NH₂)-DPhg-DTrp-Lys(CBO)-Tyr(Bzl)-Phe-],together with 190 mg DCCI and 60 mg N-hydroxysuccinimide are added. Theresulting suspension is kept at room temperature for 72 hours. Afterfiltration, the solvent is removed under reduced pressure and theremaining crude is purified on silica gel (DCM/MeOH/HOAc₅₀%8/2/0.25-->7/3/1 as mobile phase).

c) For deprotection the above DOTA—conjugate is treated with 5 mltrifluoroacetic acid/thioanisole (9/1) for two hours at roomtemperature. After that the solution is poured into a mixture of 100 mldiethylether +5 ml 3N HCl/diethylether and the resulting precipitate iasisolated by filtration. Purification is performed on silica gel usingDCM/MeOH/HOAc₅₀% 7/4/2-->7/5/4 as mobile phase. Analytically pureendproduct is obtained after a desalting step using a 0.1% TFA to 0.1%TFA in 90% CH₃CN gradient on a RP₁₈-HPLC column (Spherisorb 250×4.6 mm).MH⁺: 1434.7

Compounds of formula III, e.g. Compound B, in free form or in the formof pharmaceutically acceptable salts and complexes exhibits valuablepharmacological properties as indicated in in vitro and in vivo testsand is therefore indicated for therapy.

More particularly, compounds of formula III, e.g. Compound B, exhibit aninteresting binding profile for human somatostatin receptors (hsst). 5somatostatin receptor subtypes, sst1, sst2, sst3, sst4 and sst5 havebeen cloned and characterized. hsst1, hsst2 and hsst3 and theirsequences have been disclosed by Y. Yamada et al. in Proc. Nat. Acad.Sci., 89, 251-255 (1992). hsst4 and its sequence have been disclosed byL. Rohrer et al. in Proc. Acad. Sci., 90, 4196-4200 (1993). hsst5 andits sequence have been described by R. Panetta et al. in Mol. Pharmacol.45, 417-427, 1993.

The binding assays may be carried out as disclosed hereunder usingmembranes from cell lines expressing selectively and stably hsst1,hsst2, hsst3, hsst4 or hsst5, e.g. CHO or COS cells.

Membranes are prepared according to known methods, e.g. as disclosed byC. Bruns et al. in Biochem. J., 1990, 65, page 39-44. Membranes preparedfrom hsst selective cell lines, e.g. CHO or COS cells stably expressinghsst1 or hsst2 or hsst3 or hsst4 or hsst5 are incubated in triplicate ina total volume of 300 μl at 22° C. for 30 min with increasingconcentrations of [¹²⁵I-Tyr¹¹]-SRIF-14 in 10 mmol/l Hepes buffer (pH7.6) containing 0.5% BSA. The incubation is terminated by rapidfiltration and the filters are counted in a counter. Specific binding istotal binding minus non-specific binding in the presence of 1 μmol/lsomatostatin-14. The experiments are carried out in triplicate. Theaffinity constant (K_(D)) and number of binding sites are calculatedusing appropriate statistics and graphical programs.

Compounds of formula III, e.g. Compound B, have no significant bindingaffinity in the above binding assays towards hsst1, hsst2 and hsst4, alow binding affinity towards hsst3 and a good binding affinity towardshsst5 expressed as an IC₅₀ value in the nMolar range (IC₅₀=concentrationfor half-maximal inhibition in a competition binding assay using[¹²⁵I-Tyr¹¹]-SRIF-14 as specific radioligand).

IC₅₀

hsst1 hsst2 hsst3 hsst4 hsst5 Compound B >1000 >1000 22 nM 840 nM 0.53nM

Compounds of formula III, e.g. Compound B, show GH-release inhibitingactivity as indicated by the inhibition of GH release in vitro fromcultured pituitary cells. For example, anterior pituitary glands fromadult male rats are cut into small pieces and dispersed using 0.1%trypsin in 20 mM HEPES buffer. The dispersed cells are cultured for fourdays in MEM (Gibco) supplemented with 5% fetal calf serum, 5% horseserum, 1 mM NaHCO₃, 2.5 nM dexamethasone, 2.5 mg/ml insulin and 20 U/mlPen/Strep. On the day of the experiment the attached cells are washedtwo times with Krebs-Ringer medium buffered with 20 mM HEPES andsupplemented with 5 mM glucose and 0.2% BSA. Subsequently the cells areincubated for three hours with Compound B in the presence of 3×10⁻¹⁰ Mgrowth hormone releasing factor. The amount of growth hormone releasedinto the medium is measured by RIA.

Compounds of formula III, e.g. Compound B, inhibit the release of growthhormone (GH) in rats. Compound B is administered s.c. to anaesthetizedrats. Blood is collected after decapitation 1 h after administration ofthe compound. The duration of action is estimated on the basis of theinhibition of basal GH secretion 6 h after drug treatment. Hormonelevels are measured by RIA 1 h and 6 h after treatment. The ID₅₀-valuefor the inhibition of the hormone secretion is determined graphically(log-probit) for each experiment and the resulting values are averagedlogarithmically. In this in vivo model Compound B inhibits growthhormone release.

Compounds of formula III, e.g. Compound B, are also useful in thetreatment of tumors which are hsst3 and/or hsst5 receptor positive, asindicated in proliferation tests with various cancer cell lines bearinghsst3 and/or hsst5.

Compounds of formula III, e.g. Compound B, are accordingly useful forthe prevention or treatment of disorders with an aetiology comprising orassociated with the presence or activation of hsst3 and/or hsst5, e.g.disorders or diseases associated with excess GH-secretion e.g. in thetreatment of acromegaly or for the treatment of malignant cellproliferative diseases, e.g. cancer tumors bearing hsst3 and/or hsst5,e.g. as disclosed hereunder for the complexed conjugated Compound B.

For all the above indications the required dosage will of course varydepending upon, for example, the host, the mode of administration andthe severity of the condition to be treated. In general, however,satisfactory results are obtained by administration in the order of from1 μg to 0.7 mg/kg/day of compound of formula III, e.g. Compound B. Anindicated daily dosage for patients is in the range from about 2 μg toabout 50 mg, preferably about 0.01 to about 40 mg, e.g. about 0.01 toabout 3 mg s.c. of the compound conveniently administered in divideddoses up to 3 times a day in unit dosage form containing for examplefrom about 0.5 μg to about 25 mg, e.g. from about 2 μg to 20 mg, forexample from 2 μg to 1.5 mg of compound of formula III, e.g. Compound B.

The compounds of formula III, e.g. Compound B may be administered infree form or in pharmaceutically acceptable salt form or complexes. Suchsalts and complexes may be prepared in conventional manner and exhibitthe same order of activity as the free compound. The present inventionalso provides a pharmaceutical composition comprising a compound offormula III, e.g. Compound B, in free base form or in pharmaceuticallyacceptable salt form or complex form, together with one or morepharmaceutically acceptable diluent or carrier. Such compositions may beformulated in conventional manner. The compounds of formula III, e.g.Compound B may also be administered in sustained release form, e.g. inthe form of implants, microcapsules, microspheres or nanospherescomprising e.g. a biodegradable polymer or copolymer, in the form of aliposomal formulation, or in the form of an autogel, e.g. a solid orsemi-solid composition capable of forming a gel after interaction withpatient's body fluids.

The compounds of formula III, e.g. Compound B, or a pharmaceuticallyacceptable salt or complex thereof may be administered by anyconventional route, for example parenterally e.g. in form of injectablesolutions or suspensions (including e.g. the sustained release form asindicated above), orally using a conventional absorption enhancer, in anasal or a suppository form or topically, e.g. in the form of anophthalmic liquid, gel, oinment or suspension preparation, e.g aliposomal, microsphere or nanosphere formulation, e.g. for instillationor subconjunctival or intra- or peri-ocular injections.

In accordance with the foregoing the present invention further provides:

1. a compound of formula III, e.g. Compound B, or a pharmaceuticallyacceptable salt or complex thereof for use as a pharmaceutical;

2. A method of preventing or treating diseases or disorders as hereinindicated in a subject in need of such treatment, which method comprisesadministering to said subject an effective amount of a compound offormula III, e.g. Compound B, or a pharmaceutically acceptable salt orcomplex thereof; or

3. a compound of formula III, e.g. Compound B , or a pharmaceuticallyacceptable salt or complex thereof for use in the preparation of apharmaceutical composition for use in any method as defined under 2.above.

A conjugated compound of formula III, e.g. Compound B, or apharmaceutically acceptable salt thereof is useful either as an imagingagent, e.g. visualisation of hsst3 and/or hsst5 receptor positivetissues and cells e.g. hsst3 and/or hsst5 receptor positive tumors andmetastases, inflammatory or autoimmune disorders exhibiting somatostatinreceptors, tuberculosis or organ rejection after transplantation, whencomplexed with a detectable element, e.g. a γ- or positron-emittingnuclide, a fluorescent metal ion or a paramagnetic ion, e.g. ¹¹¹In,¹⁶¹Tb, ¹⁷⁷Lu, ⁸⁶Y, ⁶⁸Ga Eu³⁺, Gd³⁺, Fe³⁺, Mn²⁺ or Cr²⁺, or as aradiopharmaceutical for the treatment in vivo of hsst3 and/or hsst5receptor positive tumors and metastases, rheumatoid arthritis and severeinflammation conditions when complexed with an α- or β- emitting nuclideor a nuclide with Auger-e⁻-cascades, e.g. 90Y, ¹⁶¹Tb, ¹⁷⁷Lu, ²¹¹At,²¹³Bi or ²⁰¹Tl, as indicated by standard tests.

In particular, it is observed that the conjugated Compound A binds tosomatostatin receptors with pKi values of from about 8 to 10. Compoundof Example 10 complexed with e.g. ¹¹¹In, ⁸⁸Y, ⁹⁰Y or ¹⁷⁷Lu binds in thenM range to the respective sst sub-types in accordance with the bindingprofile of Compound B.

The affinity of a conjugated compound of formula III, e.g. conjugatedCompound B, and its complexes for hsst3 and/or hsst5 receptors can alsobe shown by in vivo testing, according to standard test methods, e.g. asdisclosed in GB-A-2,225,579. For example the compound of Example 10complexed with e.g. ¹¹¹In, ⁸⁸Y, ⁹⁰Y or ¹⁷⁷Lu, gives a significant tumoraccumulation 4 hours after injection into mice or rats bearing anexocrine pancreatic tumor expressing hsst5 receptors.

After administration of a conjugated compound of formula III, e.g.conjugated Compound B, in complexed form, e.g. radiolabelled with ¹¹¹In,¹⁷⁷Lu, ⁸⁶Y or ¹⁶¹Tb, at a dosage of from 1 to 5 μg/kg labelled with 0.1to 5 mCi radionuclide, preferably 0.1 to 2 mCi, the tumor site becomesdetectable.

The conjugated compound of formula III, e.g. conjugated Compound B, whenradiolabelled with an α- or β-emitting radionuclide or a nuclide withAuger-e⁻-cascades exhibits an antiproliferative and/or cytotoxic effecton tumor cells bearing hsst3 and/or hsst5 receptors, e.g. as indicatedin nude mice tests.

Nude mice are inoculated with hsst5 bearing tumor cells. When tumorshave reached a volume of 1 to 2 cm³ animals are randomized into controland treatment groups. A conjugated compound of formula III, e.g.conjugated Compound B in complexed form is administered by i.p. or i.v.injections. Doses up to 40 mCi/kg are given per mouse. The size of thetumors is determined with a caliper as disclosed above. For statisticalcalculations Student's t-test is applied. In this test, transient tumorshrinkage is observed after one week and tumor growth is delayed for twoweeks upon a single application of the compound of Example 10 complexedwith ⁹⁰Y or ¹⁷⁷Lu. In contrast the control groups showed continuoustumor growth with a volume doubling time of about seven days.

Accordingly, in a series of specific or alternative embodiments, thepresent invention also provides:

4. Use of a conjugated compound of formula III, e.g. conjugated CompoundB, complexed with a detectable element for in vivo detection of hsst3and/or hsst5 positive cells and tissues, e.g. hsst3 or hsst5 positivetumors and metastasis, in a subject and recording the localisation ofthe receptors targeted by said complex;

5. A method for in vivo detection of hsst3 and/or hsst5 positive tissuesand cells, e.g. hsst3 or hsst5 positive tumors and metastasis, in asubject comprising administering to said subject a conjugated compoundof formula III, e.g. conjugated Compound B, complexed with a detectableelement, or a pharmaceutically acceptable salt form, and recording thelocalization of the receptors targeted by said complex.

The conjugated compound of formula III, e.g. conjugated Compound B, incomplexed form for use as an imaging agent may be administered e.g.intravenously, e.g. in the form of injectable solutions or suspensions,preferably in the form of a single injection. The radiolabelling maypreferably be performed shortly before administration to a subject.

In animals an indicated dosage range may be from 0.01 to 1 μg/kg of aconjugated compound of formula III, e.g. conjugated Compound B,complexed with 0.02 to 0.5 mCi γ-emitting radionuclide. In largermammals, for example humans, an indicated dosage range may be from 1 to100 μg/m² conjugated Compound B complexed e.g. with 1 to 100 mCi/m²detectable element, e.g. ¹¹¹In, ⁸⁶Y or 177Lu.

6. Use of a conjugated compound of formula Ill, e.g. conjugated CompoundB, complexed with an α- or β-emitting nuclide or a nuclide withAuger-e⁻-cascades, for in vivo treatment of hsst3 and/or hsst5 positivetumors and metastases.

7. A method for in vivo treatment of hsst3 and/or hsst5 positive tumorsand metastases, e.g. for treating invasiveness of such tumors orsymptoms associated with such tumor growth, in a subject in need of suchtreatment which comprises administering to said subject atherapeutically effective amount of a conjugated compound of formulaIII, e.g. conjugated Compound B, complexed with an α- or β-emittingnuclide or a nuclide with Auger-e^(—)cascades.

8. Use of a conjugated compound of formula III, e.g. conjugated CompoundB, or a pharmaceutically acceptable salt thereof in the manufacture ofan imaging agent or a radiopharmaceutical composition.

Dosages employed in practising the radiotherapeutic use of the presentinvention will of course vary depending e.g. on the particular conditionto be treated, for example the known radiotoxicity to normal organsexpressing hsst5, the volume of the tumor and the therapy desired. Ingeneral, the dose is calculated on the basis of pharmacokinetik andradioactivity distribution data obtained in to healthy organs and basedon the observed target uptake. A β-emitting complex of a conjugatedcompound of formula III, e.g. conjugated Compound B, may be administeredrepeatedly e.g. over a period of 1 to 3 months.

In animals an indicated dosage range may be from 20 to 100 μg/kgconjugated compound of formula III, e.g. conjugated Compound B,complexed with 15 to 70 mCi of an α- or β-emitting nuclide or a nuclidewith Auger-e^(—)cascades, e.g. ⁹⁰Y, ¹⁷⁷Lu or ¹⁶¹Tb. In larger mammals,for example humans, an indicated dosage range may be from 1 to 100 μg/m²conjugated compound of formula III, e.g. conjugated Compound B,complexed e.g. with 1 to 100 mCi/m² of an α- or β-emitting nuclide or anuclide with Auger-e⁻-cascades, e.g. ⁹⁰Y, ¹⁷⁷Lu or 161Tb.

A conjugated compound of formula III, e.g. conjugated Compound B, incomplexed form for use as a radiotherapeutic agent may be administeredby any conventional route, e.g. intravenously, e.g. in the form ofinjectable solutions. It may also be administered advantageously byinfusion, e.g. an infusion over 15 to 60 min. Depending on the site ofthe tumor, it may be administered as close as possible to the tumorsite, e.g. by means of a catheter. The present invention also provides apharmaceutical composition comprising a conjugated compound of formulaIII, e.g. conjugated Compound B, in free base form or inpharmaceutically acceptable salt form or complexed with a detectable orradiotherapeutic agent, together with one or more pharmaceuticallyacceptable diluent or carrier.

A compound of formula III or a conjugated compound of formula III, e.g.Compound B or the conjugated Compound B, in complexed form may besuitable for imaging or treating hsst3 and/or hsst5 expressing oraccumulating such as pituitary tumors, e.g. adenomas or prolactinomas,gastro-enteropancreatic tumors, carcinoids, central nervous system,breast, prostatic (including advanced hormone-refractory prostatecancer), ovarian or colonic tumours, small cell lung cancer, malignantbowel obstruction, paragangliomas, kidney cancer, skin cancer,neuroblastomas, pheochromocytomas, medullary thyroid carcinomas,myelomas, lymphomas, Hodgkins and non-Hodgkins lymphomas, bone tumoursand metastases thereof, as well as autoimmune or inflammatory disorders,e.g. rheumatoid arthritis, Graves disease or other inflammatory eyediseases.

According to a further aspect of the invention, there is provided apharmaceutical composition comprising a conjugated compound of formulaIII, e.g. conjugated Compound B, or a complex thereof together with oneor more pharmaceutically acceptable carriers or diluents therefor. Suchcompositions may be manufactured in conventional manner and may bepresented, e.g. for imaging, in form of a kit comprising two separatedosages, one being the radionuclide and the other the conjugatedcompound of formula III, e.g. conjugated Compound B, with instructionsfor mixing them. For radiotherapy, the conjugated compound of formulaIII, e.g. conjugated Compound B, in complexed form may preferably be inthe form of a hot liquid formulation.

A compound of formula III optionally conjugated, e.g. Compound B or aconjugated Compound B, in complexed form may be administered as the soleactive ingredient or in conjuction with, e.g. as an adjuvant to, otherdrugs. For example, a compound of formula III, e.g. Compound B, may beused in combination with an immunosuppressive agent, e.g. a calcineurininhibitor, e.g. cyclosporin A, Isa Tx247 or FK 506; a mTOR inhibitor,e.g. rapamycin, CC779, ABT578 or 40-O-(2-hydroxyethyl)-rapamycin; anascomycin having immunosuppressive properties, e.g. ABT-281, ASM981,etc.; corticosteroids; cyclo-phosphamide; azathioprene; methotrexate;leflunomide; mizoribine; mycophenolic acid or a salt thereof, e.g.Myfortic^(R); mycophenolate mofetil; 15-deoxyspergualine or animmunosuppressive homologue, analogue or derivative thereof; a S1Preceptor agonist, e.g. FTY720; immunosuppressive monoclonal antibodies,e.g., monoclonal antibodies to leukocyte receptors, e.g., MHC, CD2, CD3,CD4, CD7, CD8, CD25, CD28, CD40, CD45, CD58, CD80, CD86 or to theirligands; other immunomodulatory compounds, e.g. a recombinant bindingmolecule having at least a portion of the extracellular domain of CTLA4or a mutant thereof, e.g. an at least extracellular portion of CTLA4 ora mutant thereof joined to a non-CTLA4 protein sequence, e.g. CTLA4Ig(for ex. designated ATCC 68629) or a mutant thereof, e.g. LEA29Y;adhesion molecule inhibitors, e.g. LFA-1 antagonists, ICAM-1 or −3antagonists, VCAM-4 antagonists or VLA-4 antagonists. A compound offormula III, e.g. Compound B may also be used in combination with ananti-inflammatory agent, a GH secretagogue receptor modulating agent,e.g. ghrelin or hexarelin, a GH receptor antagonist, e.g. pegvisomant,

A compound of formula III optionally conjugated, e.g. Compound B or aconjugated Compound B, in complexed form may also be used in combinationwith an antiproliferative agent, e.g. a chemotherapeutic drug, e.g.paclitaxel, gemcitabine, cisplatinum, doxorubicin, 5-fluorouracil ortaxol, a hormonal agent or antagonist, e.g. an anti-androgen ormitoxantrone (especially in the case of prostate cancer), or anantiestrogen, like letrozole (especially in the case of breast cancer),an antimetabolite, a plant alkaloid, a biological response modifier,preferably a lymphokine or interferons, an inhibitor of protein tyrosinekinase and/or serine/threonine kinase, or an agent with other or unknownmechanism of action, e.g. any epothilone or epothilone derivative, or amTOR inhibitor, e.g. as indicated above.

Where a compound of formula III optionally conjugated, e.g. Compound Bor a conjugated Compound B, in complexed form is administered inconjunction with another drug, dosages of the co-administered drug willof course vary depending on the type of co-drug employed, on thespecific drug employed, on the condition to be treated, and so forth.The terms “co-administration” or “combined administration” or the likeas utilized herein are meant to encompass administration of the selectedtherapeutic agents to a single patient, and are intended to includetreatment regimens in which the agents are not necessarily administeredby the same route of administration or at the same time.

In accordance with the foregoing the present invention provides in a yetfurther aspect:

9. A pharmaceutical combination comprising a) a first agent which is acompound of formula III optionally conjugated, e.g. Compound B or aconjugated Compound B, in complexed form and b) a co-agent, e.g. asdefined above.

10. A method as defined above comprising co-administration, e.g.concomitantly or in sequence, of a therapeutically effective amount of acompound of formula III optionally conjugated, e.g. Compound B or aconjugated Compound B in complexed form, and a second drug substance,said second drug substance being, e.g. as indicated above.

1. A liquid formulation for parenteral administration buffered at a pHof about 4 to about 4.5 and comprising as active ingredientcyclo[{4-(NH₂—C₂H₄—NH—CO—O-)Pro}-Phg-DTrp-Lys-Tyr(4-Bzl)-Phe] or apharmaceutically acceptable salt thereof.
 2. A liquid formulationaccording to claim 1 wherein the composition is buffered by anacetate/acetic acid, lactate/lactic acid, or Glycin/HCl buffer.